We propose a novel concept for the aerosolized gene therapy of the pulmonary manifestations of CF. A gene delivery system will be constructed consisting of artificial viral envelopes (virosomes) which will mimic the strategy adopted by respiratory syncytial virus (RSV) to target and penetrate respiratory cells. Encapsulated within the RSV virosome will be the CFTR CDNA in a plasmid expression vector driven by a strong promoter. The validity of this concept will be explored using the following specific aims: 1. Develop the technology for a CFTR gene delivery system targeted to respiratory cells by: a) Designing virosome carriers which effectively target and penetrate respiratory epithelial cells from patients with CF. To achieve this, alterations in the composition of the virosome will be made and the kinetics of virosome entry into CF cells measured using a fluorometric fusion assay. b) Testing the virosome as a targeted DNA delivery system in animals by harvesting the lungs, liver, and kidneys following aerosol exposure and measuring organ specificity, timing of expression, and cellular site of expression of the marker gene; and c) Constructing a plasmid expression vector (pCMV) containing the CDNA for human CFTR. 2. Utilizing RSV virosomes to deliver the CFTR-plasmid construct to the cytoplasm of respiratory cells: a) Demonstrate functional correction of chloride transport in CF bronchial epithelial cells using the video-based SPQ fluorescence assay of C1 transport, the whole-cell patch-clamp technique, and indirect immunofluorescence; and b) Effectively target, deliver, and express the gene in respiratory epithelial cells following aerosol delivery of the virosome to animals. CFTR expression will be delineated utilizing in situ hybridization to determine cell localization of CFTR, Northern analysis for detection of message, immunohistochemical staining for the CFTR protein, and measurement of transport function of tracheal epithelial cells harvested at the time of sacrifice of the animal. 3. Evaluate the potential toxic effects of CFTR-containing virosomes on respiratory structure and function by delineating the consequences of repeated instillations of the virosome in the developing lung. In particular, physiologic measures of airway function including evaluation for airway sensitization to foreign protein, detailed morphometric analysis, and assessment of alveolar macrophage function will be made in lambs given monthly intratracheal instillations of virosomes from infancy to adulthood. 4. Demonstrate effective targeting of the respiratory epithelium and subsequent correction of the CF defect using the RSV virosome delivery system and the pCMV4 CFTR expression vector in subjects with CF about to undergo lobectomy or lung transplant. Efficacy will be assessed by the SPQ fluorescence assay of C1 transport and in situ hybridization.